title: Functional identification of hyoscyamine 6β-hydroxylase from Anisodus acutangulus and overproduction of scopolamine in genetically-engineered Escherichia coli.
abstract: Hyoscyamine 6β-hydroxylase (H6H; EC 1.14.11.11) converts hyoscyamine to scopolamine in the last step of scopolamine biosynthetic pathway. The gene encoding H6H in Anisodus acutangulus was cloned and expressed in Escherichia coli and the recombinant proteins fused with His-tag or GST-tag at its N-terminal were purified and then confirmed by Western bolt analysis. The biofunctional assay revealed that the His-AaH6H and GST-AaH6H converted hyoscyamine (40 mg/l) to scopolamine at 32 and 31 mg/l, respectively. This is the first report on AaH6H expression, purification and functional characterization facilitates further genetic improvement of scopolamine yield in A. acutangulus.
authors.affiliation: Laboratory of Plant Biotechnology, College of Life and Environment Sciences, Shanghai Normal University, Shanghai, People's Republic of China. gykai@shnu.edu.cn
authors.forename: Guoyin
authors.initials: G
authors.lastname: Kai
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Liu
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Wang
authors.forename: Sheng
authors.initials: S
authors.lastname: Yang
authors.forename: Xueqing
authors.initials: X
authors.lastname: Fu
authors.forename: Xiuqin
authors.initials: X
authors.lastname: Luo
authors.forename: Pan
authors.initials: P
authors.lastname: Liao
mesh_terms: D001285:Atropine / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D053858:Metabolic Networks and Pathways / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008954:Models, Biological; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012601:Scopolamine / Q000378:metabolism*; D019657:Solanaceae / Q000201:enzymology*
chemical_list: D011994:Recombinant Proteins; D001285:Atropine; D012601:Scopolamine; D006899:Mixed Function Oxygenases; C052273:hyoscyamine (6S)-dioxygenase